Renal excretion of NH4+ is critical for acid-base homeostasis, normally accounting for at least two-thirds of daily net acid excretion in the urine. Recent studies have identified new non- erythroid Rh proteins that are related to the NH4+ transporters in non-mammalian species. Normal acid-base balance is essential for health and prevention of progression of chronic kidney disease. The main purpose of this proposal is to determine the functional and structural properties of renal Rh glycoproteins with respect to NH3/NH4+ transport and CO2 transport which is also critical for acid-base homeostasis. Among the properties to be explored are their critical sensitivity to pH; a potential role as transporters of gases and the possibility that they serve dual roles as NH4+ and NH3 transporters. Specifically we will address the following aims: 1) Transport characteristics of Rh glycoproteins: The main hypothesis of this aim is that Rhbg and Rhcg are transporters of NH4+ (and possibly NH3). 2) Structure-function characteristics of Rh glycoproteins: Site-directed mutagenesis of critical residues & functional assays will be conducted to determine why different Rh glycoproteins transport NH3/NH4+ differently. 3) Transport of NH3 and CO2 by Rh glycoproteins: We will determine if Rh proteins transport NH3 or CO2. This property is novel & unique in that very few membrane proteins have been identified as mediators of gas transport. Our studies employ a combination of techniques (pHi studies, voltage clamp experiments, site-directed mutagenesis) that should clearly establish whether Rh proteins act as NH3 or CO2 or NH4+ transporters and the characteristics of this transport. This work relies on in vitro experiments because of the difficulties inherent in characterizing NH3/NH4+ transport. The proposed work is substantial, but our ultimate goals are to understand the physiologic significance and regulation of these processes in the kidney. PUBLIC HEALTH RELEVANCE: Acid-base disorders are common in Veterans particularly during chronic kidney disease, diabetes, and hypertension. The present application aims to understand the kidney functions involved in acid-base homeostasis so that these disorders can be diagnosed and treated appropriately. The detailed aspects being studied are proteins called Rh proteins that regulate ammonia excretion in the urine, the primary mechanism that kidneys maintain acid-base balance.